Elsevier

Neuroscience

Volume 343, 20 February 2017, Pages 322-336
Neuroscience

Novel genes in brain tissues of EAE-induced normal and obese mice: Upregulation of metal ion-binding protein genes in obese-EAE mice

https://doi.org/10.1016/j.neuroscience.2016.12.002Get rights and content

Highlights

  • An aggravated EAE was observed in HFD-induced obese mice compared with ND-mice.

  • Significantly upregulated genes in EAE were related to immune response, antigen presentation, complement activation, etc.

  • C4b, Psmb8, Ly86, Ms4a6d, Cul9, and Mast2 are novel genes upregulated in both ND- and HFD-EAE mice.

  • Cul9 and Mast2 genes of metal ion-binding proteins were upregulated in HFD-EAE mice than in ND-EAE mice.

Abstract

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory autoimmune disease of the central nervous system resulting from degeneration of the myelin sheath. This study is aimed to identify differentially expressed genes (DEGs) in the brain of EAE-induced normal diet (ND) mice and high-fat diet (HFD)-induced obese mice, and to identify novel genes responsible for elucidating the mechanism of the disease. Purified mRNA samples from the brain tissue were analyzed for gene microarray and validated by real-time RT-PCR. DEGs were identified if significant changes greater than 1.5-fold or less than 0.66-fold were observed (p < 0.05). Pathway construction and functional categorization were performed using the Kyoto encyclopedia of genes and genomes pathways and gene ontology (GO) analysis. HFD-EAE mice showed more severe disease symptoms than ND-EAE mice. From GO study, fold changes of HFD-EAE to ND-EAE genes indicated that the genes were significantly associated to the pathways related with the immune response, antigen presentation, and complement activation. The genes related with metal ion-binding proteins were upregulated in HFD-EAE and ND-EAE mice. Upregulation of Cul9, Mast2, and C4b expression is significantly higher in HFD-EAE mice than ND-EAE mice. Cul9, Mast2, C4b, Psmb8, Ly86, and Ms4a6d were significantly upregulated in both ND- and HFD-EAE mice. Fcgr4, S3-12, Gca, and Zdhhc4 were upregulated only in ND-EAE, and Xlr4b was upregulated only in HFD-EAE mice. And significant upregulated genes of metal ion-binding proteins (Cul9 and Mast2) were observed in HFD-EAE mice.

Introduction

Multiple sclerosis (MS) is a human autoimmune inflammatory disorder resulting from demyelination of neurons in the central nervous system (CNS). A notable event of MS is the transfer of immune cells into the CNS through the blood–brain barrier (BBB). The inflammatory cells then interact with astrocytes and microglia and induce demyelination. Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease mediated by T cells and is most commonly used as an animal model of MS. Although T-helper type 1 (Th1) cells are recognized as the main effector T cells for autoimmune inflammation, recent studies have also focused on CD4 + Th17 cells that secrete interleukin 17 (IL-17) in different chronic inflammatory pathological conditions including EAE (Fletcher et al., 2010).

Obesity is a metabolic disorder associated with a low grade of inflammation in the periphery. Recent studies have confirmed the association of brain inflammation in a rodent model of diet-induced obesity (Kanneganti and Dixit, 2012). A positive correlation between peripheral inflammation and cognitive decline was also reported in obese subjects (Sellbom and Gunstad, 2012). Diet-induced obese mice were found to have exacerbated 2,4,6-trinitrobenzene sulfonic acid-induced colitis and EAE disease compared with normal diet (ND) mice (Winer et al., 2009). Genomic factors are closely related to MS as monozygotic twins display higher concordance compared with dizygotic twins, and the relatives of MS patients have a 15% chance of being affected (Tajouri et al., 2007). However, the precise mechanism of the susceptibility of HFD-induced obese mice to EAE is still undefined.

The revolutionary approach of microarray-based genomic technology has benefited the genetic analysis of the complex biology of human diseases. Both the brain and spinal cord were studied extensively for functional research on drug testing and mechanism studies of EAE. Transcriptome of the choroid plexus, a part of BBB, demonstrated the altered expression of adhesion molecules, cytokines, and chemokines. The lipocalin-2 was the most upregulated gene in EAE mice. MS brain (astrocytes) also had the increased expression of lipocalin-2 (Marques et al., 2012). The key enzyme in leukotriene biosynthesis, 5-lipoxygenase, was upregulated and identified in MS lesions and in EAE brain by comparing the gene expression analysis using a cDNA microarray consisting of 2978 human genes (Whitney et al., 2001). The putative genes for susceptibility to EAE were also revealed by an oligonucleotide microarray of inflamed spinal cords of EAE at the onset and the peak, and 100 genes were found to be consistent with differential regulation throughout the disease (Ibrahim et al., 2001b).

Gene expression studies in the spinal cord of myelin basic protein-treated female Lewis rats confirmed the mostly differentially regulated genes associated with induction of the disease, e.g., genes associated with antigen presentation, chemotaxis, cell signaling, and infiltration of immune cells. However, there were also genes associated with processes involved in the resolution of the disease, e.g., apoptosis of immune filtrating cells, oligodendrocyte precursor cells, etc. (Inglis et al., 2012). The genetic susceptibility analysis of EAE was performed in the CNS of different mouse strains. However, the responsive genes in the brain’s frontal cortex related to EAE were not revealed so far in normal and HFD-induced obese mice.

In current study, we demonstrated the differential gene expression in the brain tissue of myelin oligodendrocyte glycoprotein 35-55 (MOG3555)-induced ND- and HFD-EAE mice. Although there are dissimilarities between EAE and MS, EAE still represents the closely matched animal model of MS. Moreover, the patterns of CNS lesions in MOG3555-induced EAE mice are identical in spinal cord and brain tissues (Kuerten et al., 2007). Currently, EAE is the most widely used model to study for functional approach toward mechanisms, drug testing, and treatment strategy of MS. In that sense, analysis with EAE brain tissues might give an idea of how real responses are generated in MS condition (Zeis et al., 2008). The identification of the gene pools involved in a severely diseased condition is emphasized. Therefore, brain samples from both ND and HFD groups were collected at day 30 post-immunization after the peak disease clinical scores had been confirmed. Differentially expressed genes (DEGs) were revealed in both ND- and HFD-EAE mice compared with their respective controls. Upregulated genes related to increased disease severity in HFD-EAE mice were also validated.

Section snippets

Materials

High-fat diets (HFDs) (60% calories from fat) were purchased from Research Diet Inc. (NJ, USA). Immunization peptide MOG35–55 was synthesized from A&Pep (Chungnam, Korea) and complete Freund’s adjuvant was purchased from Difco Laboratories (MI, USA). Trizol RNA extraction reagent was purchased from Invitrogen™ (CA, USA). Genomic DNA was removed by the RNase-free DNase set, and RNA purification was performed using the RNeasy mini kit Qiagen (Hilden, Germany). SYBR Green supermix kit was from

Aggravated EAE in obese mice

The HFD-induced body weight of mice increased by 40% over the weight of ND mice (37.6 g vs. 27.8 g) after ten weeks (Fig. 1A). EAE was induced in both ND mice and HFD-induced obese mice by immunization with the MOG33-35 peptide. The changes in body weight at day 30 and the cumulative body weight changes (Fig. 1B) were significant in EAE mice but not in control mice. At day 30, the body weight of ND-EAE mice was 15% lower than that of ND-CON. However, in the case of HFD-EAE, the decrement was 37%

Discussion

MS is a demyelinating disease that primarily affects the motor and sensory functions of the brain (Noseworthy et al., 2000). Although demyelination and inflammation of the white matter result in MS pathology, the gray matters ranging from cortex to other deep regions have also been shown to be affected (Prins et al., 2013). In addition to genetic factors (Sawcer et al., 2011), environmental factors also modulate autoimmune diseases such as MS and type-1 diabetes mellitus. Diet is regarded as

Conclusion

HFD-EAE mice showed more severe disease symptoms than ND-EAE mice. The novel upregulated genes of Fcgr4, C4b, Psmb8, Ly86, Xlr4b, S3-12, and Ms4a6d were observed in EAE mice. The metal ion-binding genes Cul9, Gca, Zdhhc4, and Mast2 were also upregulated in EAE mice. By real-time RT-PCR were validated the upregulated genes of Cul9, Mast2, C4b, Psmb8, Ly86, and Ms4a6d in both ND- and HFD-EAE mice; of Fcgr4, S3-12, Gca, and Zdhhc4 only in ND-EAE mice; and of Xlr4b only in HFD-EAE mice. And the

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgments

This research was supported by Public Welfare & Safety Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant number 2011-0020952), and also partly supported by the research fund from Korea Institute of Science and Technology (2V04070, 2V04620).

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